Invasive fungal infections (IFIs) are associated with high rates of morbidity and mortality and pose a serious health concern for severely immunocompromised patients. Infections caused by pathogenic Candida species are especially prevalent, affecting nearly one in fifty intensive care unit patients and causing nearly 10% of all ICU-acquired bloodstream infections in North America. Preventing these deadly infections is hindered by the continued development of antifungal drug resistance in these pathogenic species. Multidrug resistance is primarily acquired by transcriptional upregulation of membrane efflux pumps, and by overexpression of drug target genes within the ergosterol biosynthesis pathway. Members of the fungal-specific zinc cluster family of transcription factors (TFs) are primarily responsible for the upregulation of these efflux pumps and ergosterol biosynthesis enzymes and thereby mediating pleiotropic drug resistance (PDR) in yeast. The identification of small molecule drugs that target these TFs, and potentially inhibit or alter their DNA-binding activity, and thus reduce their ability to upregulate target genes involved in PDR could result in significant improvements to current antifungal drug therapies. The overarching goal of this project is to identify small molecule inhibitors of PDR TFs involved in antifungal drug resistance in Saccharomyces cerevisiae as well as the pathogenic species Candida albicans and Candida glabrata. Libraries of diverse compounds will be screened by small molecule microarrays (SMMs) to identify those compounds capable of direct interaction with these TFs. Protein binding microarrays (PBMs) will be used to characterize these compounds' ability to alter or inhibit zinc cluster TF DNA binding activity. In parallel, in vivo drug susceptibility tests will be performed to examine the in vivo effects of the small molecule 'hits' from the SMM screen on S. cerevisiae, C. albicans and C. glabrata survival and susceptibility to these compounds; compounds will be tested in vivo both individually and in combination with each other as well as with existing antifungal drugs. Successful completion of this study will allow for the development of a new methodology for identifying TF inhibitors capable of preventing sequence-specific DNA binding, and will expand the role of TFs as potential drug targets. Ultimately, small molecules identified in this project may serve as lead compounds for development of improved antifungal drugs in the treatment of invasive fungal infections. PUBLIC HEALTH RELEVANCE: The development of improved antifungal drugs and the expansion and discovery of novel drug targets is one of the most important needs for the future treatment of invasive fungal infections. This project is focused on identifying novel small molecules that target several key sequence-specific transcription factors controlling pleiotropic drug resistance, potentially by inhibiting or altering their DNA-binding activity, and thus reduce their ability to upregulate multidrug efflux pumps and ergosterol biosynthesis genes in the species Saccharomyces cerevisiae, Candida albicans and Candida glabrata. Successful completion of this research will: (a) develop a new methodology for identifying inhibitors of sequence-specific DNA binding by transcription factors; (b) expand the general role of transcription factors as potential drug targets; and (c) identify potential lead compounds for development of novel antifungal drugs in the treatment of invasive fungal infections.